Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Evidence suggests that medial preoptic area (MPOA) neurones containing gamma-aminobutyric acid (GABA) are modulated directly by oestrogen. We have used an alkaline phosphatase-labelled antisense oligonucleotide probe to examine glutamic acid decarboxylase67 (GAD) mRNA expression within individual cells of the MPOA, diagonal band of Broca (DBB) and parietal cortex in rats killed at noon on each day of the oestrous cycle and after ovariectomy (n = 4-5). As a fall in extracellular GABA concentrations occurs in the MPOA on the afternoon of proestrus, the GAD67 mRNA content of cells was also examined in proestrous rats at 15:00h immediately prior to the preovulatory luteinising hormone (LH) surge. The MPOA was found to have an intermediate number of GAD67 mRNA-containing cells compared with the DBB and cortex (P less than 0.01) but expressed the lowest mean hybridisation signal (P less than 0.01). The parietal cortex had significantly fewer (P less than 0.01) GAD mRNA-containing cells than either the MPOA or DBB but these contained higher mean density of signal (P less than 0.01). The hybridisation signal for GAD mRNA was abolished by either ribonuclease pre-treatment or the use of excess non-labelled probe. No significant (P greater than 0.05) differences in GAD67 mRNA were detected in animals killed at noon throughout the oestrous cycle or after ovariectomy. On the afternoon of proestrus (15:00h) there was a significant 40% reduction in mean GAD67 mRNA content within cells of only the MPOA compared with noon (P less than 0.05). The numbers of cells in the MPOA expressing GAD67 mRNA were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression of glutamic acid decarboxylase messenger RNA in rat medial preoptic area neurones during the oestrous cycle and after ovariectomy. 132 94

Glutamic acid decarboxylase (GAD), a target of both autoantibodies and autoreactive T-cells in insulin-dependent diabetes (IDD), exists as two homologous forms, GAD65 and GAD67. GAD65 is preferentially expressed in human islets and recognized by autoantibodies in IDD, but which form primarily elicits GAD autoimmunity is unknown. GAD67 gene expression in human islets has been demonstrated only by the polymerase chain reaction. We, therefore, quantitatively compared the expression of each GAD gene in human islets and mapped the binding of autoantibodies to recombinant human GAD67 by enzyme-linked immunosorbent assay. In ribonuclease protection assays, both forms of GAD messenger RNA (mRNA) were detected in human islets, although GAD65 mRNA was 200 times more abundant than GAD67 mRNA. Immunoblotting of islets with GAD form-specific antisera revealed GAD65, but not GAD67. By in situ hybridization and immunohistochemistry, GAD65 mRNA and protein were localized to islets, predominantly, but not entirely, to beta-cells; GAD67 mRNA and protein were undetectable. Thus, although GAD67 protein expression was undetectable in human islets, the GAD67 gene is transcribed, albeit weakly. Antibodies that recognized multiple epitopes in recombinant GAD67 were found in 20% of sera from ICA positive "at risk" first degree relatives of IDD subjects and recent-onset IDD subjects. The majority of GAD67 epitopes were mapped within the mid- and C-terminal thirds of the protein, a region that is highly conserved in GAD65. Although GAD67 may share cross-reactive epitopes with GAD65, these findings do not exclude the possibility that autoimmunity to GAD arises as a consequence of the aberrant up-regulation of GAD67 in human islets.
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PMID:Glutamic acid decarboxylase-67 (GAD67): expression relative to GAD65 in human islets and mapping of autoantibody epitopes. 753 77

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease in which cytokines are thought to play an important role in beta-cell destruction and immune regulation. A major target of beta-cell autoimmunity in IDDM is the enzyme glutamate decarboxylase (GAD). We hypothesized that cytokines in the insulitis lesion modulate the synthesis of GAD. This may, in turn, modify the rate of beta-cell destruction. Accordingly we cultured rat islets in the presence and absence of cytokines, and measured synthesis of both isoforms of GAD, GAD65 and GAD67, by [35S]methionine incorporation and immunoprecipitation with a rabbit antiserum that recognizes both GAD65 and GAD67. Incubation of islets with interleukin (IL)-1 beta (1 ng/ml, 24 h), tumour necrosis factor alpha (TNF-alpha; 200 units/ml, 24 h) or interferon gamma (IFN-gamma; 500 units/ml, 72 h) significantly decreased the synthesis of both GAD65 and GAD67, but reduced neither total protein synthesis nor insulin accumulation in the medium or content. Incubation of islets for 24 h in IFN-alpha (1000 units/ml), TNF-beta (50 ng/ml), IL 2 (1000 units/ml), IL-4 (100 ng/ml), IL-6 (10 ng/ml), IL-10 (20 ng/ml), IL-12 (10 ng/ml) or transforming growth factor beta 2 (TGF-beta 2; 5 ng/ml) did not significantly alter GAD65 or GAD67 synthesis. Inhibition of GAD65 and GAD67 protein synthesis by IL-1 beta, TNF-alpha or IFN-gamma was reversed by co-incubation with the nitric oxide synthase inhibitor, NG-monomethyl arginine (NMMA). Expression of both GAD65 and GAD67 mRNA, measured by RNase protection assay, was also decreased by IL-1 beta and completely restored to baseline levels by NMMA. Thus the synthesis of both isoforms of islet GAD is selectively decreased in the presence of IL-1 beta, TNF-alpha or IFN-gamma by a NO-mediated mechanism, probably at the level of cytokine gene transcription. As GAD autoimmunity has been previously shown to have a pathogenic role in an animal model of IDDM, its inhibition by cytokines might limit the immune response, thereby regulating the rate of beta-cell destruction in IDDM.
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PMID:Cytokine regulation of glutamate decarboxylase biosynthesis in isolated rat islets of Langerhans. 876 Mar 54

gamma-Aminobutyric acid (GABA)ergic neurons terminating in the rostral hypothalamus are stimulated by testosterone. To investigate whether this action is mediated locally through androgen receptors in the rostral hypothalamus, bilateral microcannulas (28 gauge) containing the androgen receptor antagonist, hydroxyflutamide (HF), were stereotaxically implanted into the rostral medial preoptic area (rMPA) just dorsal to the major population of GnRH cell bodies. Two days later, blood samples were collected for assay of LH, and animals were killed for determination of GABAergic neuronal activity in tissue dissected from the site of the implanted cannulas. Animals were decapitated either without treatment or 60 min after inhibition of GABA degradation by aminooxyacetic acid (100 mg/kg, ip). The rate of GABA accumulation in the tissue after aminooxyacetic acid treatment was used as a measure of GABA turnover. Levels of messenger RNA for both forms of glutamic acid decarboxylase (GAD65 and GAD67), the rate-limiting enzyme responsible for GABA synthesis also were measured by a microlysate ribonuclease protection assay. LH levels were significantly increased (1.8-fold) in HF-treated animals compared with controls. In the MPA, beneath the implant cannulas, GABA turnover was significantly reduced in HF-treated rats. There was no effect of treatment in the frontal cortex, which was used as a control region. Surprisingly, levels of messenger RNA for both GAD65 and GAD67 were significantly increased in HF-treated rats. The results indicate that GABAergic neurons terminating in the rostral hypothalamus are tonically stimulated by testosterone acting by means of androgen receptors localized in this region. These findings support the working hypothesis that androgen-sensitive GABAergic neurons in the rMPA mediate the negative feedback action of testosterone on GnRH secretion in the male rat.
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PMID:Antiandrogen microimplants into the rostral medial preoptic area decrease gamma-aminobutyric acidergic neuronal activity and increase luteinizing hormone secretion in the intact male rat. 882 73

There is considerable evidence that GABAergic neurons play an important role in the regulation of gonadotropin-releasing hormone (GnRH) secretion, and that these neurons may mediate the feedback actions of gonadal steroids on GnRH neurons. The aim of the present study was to investigate whether endogenous changes in ovarian steroid secretion during the estrous cycle influenced GABAergic neuronal activity in the preoptic region of the hypothalamus, and in other steroid-sensitive brain regions. Intact, adult female rats were sacrificed at various times during the days of metestrus or proestrus. GABAergic neuronal activity was estimated by measuring the rate of accumulation of GABA in microdissected brain regions after pharmacological inhibition of GABA degradation. Concentrations of mRNA for both forms of glutamic acid decarboxylase (GAD65 and GAD67) were quantified in microdissected brain regions by a microlysate ribonuclease protection assay. In the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis (DBB(ovlt)), GABAergic neuronal activity was significantly reduced during the afternoon of proestrus compared with the morning of either proestrus or metestrus. In the lateral septal nucleus, GABAergic neuronal activity was significantly increased in the afternoon of proestrus compared with the morning. There were no significant effects of time of day or day of estrous cycle in the medial preoptic nucleus, median eminence, ventromedial nucleus, suprachiasmatic nucleus, medial septal nucleus, hippocampus (CA1 region), or cingulate cortex. In the DBB(ovlt), mRNA levels for both GAD65 and GAD67 were significantly reduced in the afternoon of proestrus compared with the afternoon of metestrus. By contrast, there was no change in GAD65 and GAD67 mRNA levels in the cingulate cortex at any of the times examined. These results demonstrate that GABAergic neuronal activity, and mRNA levels for both GAD65 and GAD67, are reduced in the DBB(ovlt) during the afternoon of proestrus. These results support the hypothesis that decreased GABAergic neuronal activity in this region plays a major permissive role in the generation and maintenance of the estrogen-induced LH surge.
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PMID:GABAergic neuronal activity and mRNA levels for both forms of glutamic acid decarboxylase (GAD65 and GAD67) are reduced in the diagonal band of Broca during the afternoon of proestrus. 889 Dec 47

The testicular regulation of luteinizing hormone (LH) secretion in the adult rhesus monkey is mediated by an indirect action of testosterone to decelerate pulsatile gonadotrophin releasing hormone (GnRH) release. Whether this negative feedback action of testosterone involves regulation of GnRH gene expression is unknown. Therefore, the effect of bilateral orchidectomy on hypothalamic levels of the mRNA encoding this hypophysiotropic factor was examined. The feedback action of testosterone is generally considered to be mediated through non-GnRH cells, and the present experiment provided the opportunity to also examine testicular influences on mRNAs encoding putative hypothalamic factors implicated in the testicular regulation of LH secretion. Adult male rhesus monkeys were orchidectomized (n=5) or sham-orchidectomized (n=5) and killed 6 weeks later, after a castration-induced hypersecretion of LH was established. Separate preoptic and mediobasal hypothalamus containing areas were collected, and levels of GnRH mRNA, as well as those of mRNAs encoding pro-opiomelanocortin (POMC), the gamma-aminobutyric acid (GABA) synthesizing enzymes (glutamic acid decarboxylase 65 and 67; GAD65 and GAD67, respectively), neuropeptide Y, galanin and transforming growth factor (TGF)alpha, were quantified using RNase protection assay. Values were expressed in terms of optical density relative to that of cyclophilin mRNA levels. Bilateral orchidectomy produced a significant increase in GnRH mRNA levels that was restricted to the mediobasal hypothalamus and that was associated with a significant decrease in POMC, GAD65 and GAD67 mRNA levels in this region of the hypothalamus. In contrast, neuropeptide Y, galanin and TGFalpha mRNA levels were not affected by castration. These results indicate that, in the monkey, the deceleration of pulsatile GnRH release that is imposed by the testis, and presumably mediated by testosterone, is associated with a concomitant down regulation of GnRH gene expression in the mediobasal hypothalamus. They also support the notion that this hypothalamic feedback action may be mediated by POMC-and GABA-producing neurones in the mediobasal hypothalamus.
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PMID:Effects of orchidectomy on levels of the mRNAs encoding gonadotropin-releasing hormone and other hypothalamic peptides in the adult male rhesus monkey (Macaca mulatta). 1071 12

This study examined whether changes in the levels of the messenger RNAs (mRNAs) encoding the gamma-aminobutyric acid (GABA) synthesizing enzymes, glutamate decarboxylase (GAD)65 and GAD67 and transforming growth factor-alpha (TGFalpha) in the hypothalamus are correlated with the arrest of pulsatile GnRH release during infancy in the agonadal male monkey. This experiment also provided the opportunity to examine changes in hypothalamic GnRH gene expression during this critical phase of primate development. Male rhesus monkeys were castrated at 1 week of age: four were killed 4-7 weeks after orchidectomy while pulsatile GnRH release was robust as reflected by high circulating LH levels, and four were killed at 12-15 months of age after establishing that pulsatile GnRH release had been arrested. GAD65, GAD67, TGFalpha, and GnRH mRNA levels were estimated using RNase protection assays employing homologous probes and the results were expressed relative to cyclophilin mRNA levels. GnRH peptide was measured by RIA. GAD65 and GAD67 mRNA levels in the hypothalamus of juveniles were significantly greater than those in neonatal monkeys. On the other hand, hypothalamic TGFalpha and GnRH mRNA (and peptide) levels in agonadal neonate and juvenile monkeys were indistinguishable. These results indicate that the molecular concomitants associated with bringing the hypothalamic GnRH pulse generator into check in agonadal neonatal males are not a mirror image of those previously reported at the time this neuronal network is reactivated at puberty when TGFalpha and GnRH gene expression increase and GAD65 and GAD67 mRNA levels remain unchanged. Thus, the neurobiological mechanism that reactivates pulsatile GnRH release at puberty is likely to involve more than a simple reversal of that underlying inhibition of the same network in late infancy.
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PMID:Changes in hypothalamic gene expression associated with the arrest of pulsatile gonadotropin-releasing hormone release during infancy in the agonadal male rhesus monkey (Macaca mulatta). 1096 98

Inositolphosphates and phosphatidylinositides are important second messengers. Previously p42(IP4), a protein with high affinity for both Ins(1,3,4,5)P(4) and PtdIns(3,4,5)P(3) has been characterized in our laboratory. In the present study mRNA levels of p42(IP4) were quantified during development (ages: 7, 14, 21 days and adult) by means of ribonuclease protection assay in various rat brain regions (cerebellum, cortex, striatum, thalamus, hypothalamus, olfactory bulb, hippocampus and tectum (superior and inferior colliculus)). A high level of p42(IP4) mRNA was detected in the cortex (ca. 1 pg specific RNA per microg of total RNA) which stayed highly independent of the age of the animals. In hippocampus and in the thalamus, p42(IP4) mRNA levels were comparable to those in the cortex in the first and second week postnatally, but decreased to lower levels in the adult brain. In striatum, the mRNA increased, albeit less intensely than in hippocampus and thalamus, until day 21 postnatally, and then decreased in the adult rat brain. Cerebellar p42(IP4) mRNA showed a slow increase within the first 3 weeks postnatally, and remained rather high in the adult brain. The protein expression of p42(IP4), tested within the same samples by Western blot staining, was consistent with mRNA values. For comparison, glutamic acid decarboxylase (isoforms GAD65/GAD67), an enzyme, for which some regional brain specific distribution is already known, was also examined. The mRNA levels of GAD and its developmental regulation clearly differed from that of p42(IP4). In summary, p42(IP4) expressed in several neuronal cell types, did not seem to be restricted to specific developmental stages, but the high absolute expression levels at all developmental stages indicated that p42(IP4) is a protein fundamental for neuronal functioning.
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PMID:Expression of the brain-specific membrane adapter protein p42IP4/centaurin alpha, a Ins(1,3,4,5)P4/PtdIns(3,4,5)P3 binding protein, in developing rat brain. 1269 46

The present study tested the hypothesis that estradiol reduces tissue infarction after middle cerebral artery occlusion (MCAO) in estradiol-deficient females by augmenting glutamic acid decarboxylase (GAD) expression and thus activity, leading to increases in gamma-amino-butyric acid (GABA) tissue levels. Glutamic acid decarboxylase is the principal enzyme for GABA synthesis and has two isoforms, GAD65 and GAD67, which differ in size and cellular distribution. Rats were ovariectomized 7 to 8 days before receiving no hormone, placebo, or 25 microg estradiol via subcutaneous implant 7 to 10 days before harvesting tissue in either ischemic cohorts after 2 h of MCAO (end-ischemia) or in nonischemic cohorts. Selected cortical and striatal regions were microdissected from harvested brains. GAD65/67 mRNA levels were determined by microlysate ribonuclease protection assay. End-ischemic GABA concentrations were determined by HPLC. Steroid treatment selectively decreased ischemic cortical GAD67 mRNA levels. In most brain regions evaluated, regional GABA concentrations increased with ischemia regardless of treatment. Estradiol blocked MCAO-induced increases in GABA concentration only in dorsomedial cortex. These data suggest that estradiol repletion in ischemic rat brain selectively decreases GAD67 mRNA levels but does not alter steady-state GABA concentrations. It may be that estradiol under ischemic conditions is attenuating GABA metabolism rather than enhancing synthesis or is augmenting other aspects of GABAergic transmission such as GABA transporters and receptors.
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PMID:Estradiol alters only GAD67 mRNA levels in ischemic rat brain with no consequent effects on GABA. 1609 13